Effects of genistein and equol on human and rat testicular 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities

The objective of the present study was to investigate the effects of genistein and equol on 3β-hydroxysteroid de- hydrogenase （3β-HSD） and 17β-hydroxysteroid dehydrogenase 3 （17β-HSD3） in human and rat testis microsomes. These enzymes （3β-HSD and 17β-HSD3）, along with two others （cytochrome P450 side-chain cleavage enzyme and cytochrome P450 17α-hydroxylase/17-20 lyase）, catalyze the reactions that convert the steroid cholesterol into the sex hormone testosterone. Genistein inhibited 3β-HSD activity （0.2 μmol L^-1 pregnenolone） with half-maximal inhibition or a half-maximal inhibitory concentration （IC50） of 87 ± 15 （human） and 636 ± 155 nmol L^-1 （rat）. Genistein＇s mode of action on 3β-HSD activity was competitive for the substrate pregnenolonrge and noncompetitive for the cofactor NAD＋. There was no difference in genistein＇s potency of 3β-HSD inhibition between intact rat Leydig cells and testis microsomes. In contrast to its potent inhibition of 3β-HSD, genistein had lesser effects on human and rat 17β-HSD3 （0.1 μmol L^-1 androstenedione）, with an IC50 〉 100μmol L^-1. On the other hand, equol only inhibited human 3β-HSD by 42%, and had no effect on 3β-HSD and 17β-HSD3 in rat tissues. These observations imply that the ability of soy isoflavones to regulate androgen biosynthesis in Leydig cells is due in part to action on Leydig cell 3β- HSD activity. Given the increasing intake of soy-based food products and their potential effect on blood androgen levels, these findings are greatly relevant to public health.

Carbon Chain Length Determines Inhibitory Potency of Perfluoroalkyl Sulfonic Acids on Human Placental 3β-Hydroxysteroid Dehydrogenase 1:Screening,Structure-Activity Relationship,and In Silico Analysis

Objective This study aimed to compare 9 perfluoroalkyl sulfonic acids(PFSA)with carbon chain lengths(C4–C12)to inhibit human placental 3β-hydroxysteroid dehydrogenase 1(3β-HSD1),aromatase,and rat 3β-HSD4 activities.Methods Human and rat placental 3β-HSDs activities were determined by converting pregnenolone to progesterone and progesterone secretion in JEG-3 cells was determined using HPLC/MS–MS,and human aromatase activity was determined by radioimmunoassay.Results PFSA inhibited human 3β-HSD1 structure-dependently in the order:perfluorooctanesulfonic acid(PFOS,half-maximum inhibitory concentration,IC50:9.03±4.83μmol/L)>perfluorodecanesulfonic acid(PFDS,42.52±8.99μmol/L)>perfluoroheptanesulfonic acid(PFHpS,112.6±29.39μmol/L)>perfluorobutanesulfonic acid(PFBS)=perfluoropentanesulfonic acid(PFPS)=perfluorohexanesulfonic acid(PFHxS)=perfluorododecanesulfonic acid(PFDoS)(ineffective at 100μmol/L).6:2FTS(1H,1H,2H,2H-perfluorooctanesulfonic acid)and 8:2FTS(1H,1H,2H,2H-perfluorodecanesulfonic acid)did not inhibit human 3β-HSD1.PFOS and PFHpS are mixed inhibitors,whereas PFDS is a competitive inhibitor.Moreover,1–10μmol/L PFOS and PFDS significantly reduced progesterone biosynthesis in JEG-3 cells.Docking analysis revealed that PFSA binds to the steroid-binding site of human 3β-HSD1 in a carbon chain length-dependent manner.All 100μmol/L PFSA solutions did not affect rat 3β-HSD4 and human placental aromatase activity.Conclusion Carbon chain length determines inhibitory potency of PFSA on human placental 3β-HSD1 in a V-shaped transition at PFOS(C8),with inhibitory potency of PFOS>PFDS>PFHpS>PFBS=PFPS=PFHxS=PFDoS=6:2FTS=8:2FTS.

Alcohol dehydrogenase coexisted solid-state electrochemiluminescence biosensor for detection of p53 gene

An alcohol dehydrogenase （ADH）-coexisted solidstate electrochemiluminescence （ECL） biosensor for sensitive detection of the p53 gene was developed. The electrode modified by multiwalled carbon nanotubes, Ru（bpy）]2＋3 and polypyrrole （ MWNTs-Ru （bpy） ]2＋3 -PPy ） was prepared to adsorb the ssDNA by electrostatic interactions. Then, the ssDNA recognized the gold nanoparticles （AuNPs）-labeled p53 gene and produced the AuNPs-dsDNA electrode with the AuNPs layer. The AuNPs layer adsorbed the ADH molecules for producing the ECL signal. Thus, the biosensor was based on coupling enzyme substrate reaction with solid-state ECL detection, and it displayed good sensitivity and specificity. The detection limit of the wild type p53 sequence （wtp53） is as low as 0. 1 pmol/L and the discrimination is up to 57. 1% between the wtp53 and the muted type p53 sequence （mtp53）. The amenability of this method to the analyses of p53 from normal and cancer cell lysates is demonstrated. The signal of wtp53 in the MGC-803 gastric cancer cell lysates turns out to be about 61.8% that of the wtp53 in the GES-1 normal gastric mucosal cell lysates, and the concentration of the wtp53 is found to decrease about 59 times. The method is highly complementary to enzyme-linked immunosorbent assay （ELISA）, and it holds promise for the diagnosis and management of cancer.

11β-hydroxysteroid dehydrogenase types 1 and 2. in postnatal development of rat testis： gene express,on, localization and regulation by luteinizing hormone and androgens

11β-hydroxysteroid dehydrogenase type 1 （11β-HSD1） and type 2 （11β-HSD2） are expressed in rat testis, where they regulate the local concentrations of glucocorticoids. Here, we investigated the expression and localization of 11β-HSD in rat testis during postnatal development, and the regulation of these genes by luteinizing hormone （LH） and androgens, mRNA and protein levels were analyzed by quantitative real-time-polymerase chain reaction and western blotting, respectively, in testes collected from rats at postnatal day （PND） 7, 14, 21, 35, and 90, and from rats treated with LH, 7α.methyl-19-nortestosterone （MENT） and testosterone at PND 21 and PND 90. Immunohistochemical staining was used to identify the localization of the 11β-HSD in rat testis at PND 7, 14, and 90. We found that 11β-HSD1 expression was restricted to the interstitial areas, and that its levels increased during rat testis development. In contrast, whereas 11β-HSD2 was expressed in both the interstitial areas and seminiferous tubules at PND 7, it was present only in the interstitial areas at PND 90, and its levels declined during testicular development. Moreover, 11β-HSD1 mRNA was induced by LH in both the PND 21 and 90 testes and by MENT at PND 21, whereas 11β-HSD2 mRNA was induced by testosterone and MENT in the PND 21 testis and by LH in the PND 90 testis. In conclusion, our study indicates that the 11β-HSD1 and 11β-HSD2 genes have distinct patterns of spatiotemporal expression and hormonal regulation during postnatal development of the rat testis.

Effect of artmether,hemin and Fe^(3+) on recombinant lactate dehydrogenase from Schistosoma japonicum

Objective:To explore antischitosome effects of artemether,hemin and Fe on S/LDH.Methods: Enzyme activity of rS/LDH was assayed in the standard reaction system by adding different concentration of reagents(0.00-0.10 mM artemether,0.00-0.02 mM hemin,0.00-0.50 mM Fe^(3+)). Same solvents of the each reagent were used as control.Results:There was no enzyme activity inhibition observed at 0.10 mM artemther:obivious inhibition for lactate oxidation reaction and pyruvate reduction reaction were detected at 0.002 mM and 0.004 mM of hemin,respectively: comparing with that of the control(P<0.05).The relative enzymatic activity inhibitions for pyruvate reduction reaction and lactate oxidation reaction at 0.02 mM hemin were 93.48%and 100.00%,respectively,comparing with that of the control(P<0.01):both pyruvate reduction and lactate oxidation reaction were inhibited completely at 0.50 mM Fe^(3+),comparing with that of the control(P<0.01).Conclusions:The results implied that SjLDH was not the direct molecular target of artemether.Hemin and Fe are inhibitors of SjLDH.

Cloning and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase Encoding Gene in Gracilaria/Gracilariopsis lemaneiformis

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） plays important roles in various cellular processes. A cytosolic GAPDH encoding gene （gpd） of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5＇-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues （CL to LF）, thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

Follow-up and multimodal imaging in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency

Dear Editor, The deficit of 3-hydroxyacyl-CoA dehydrogenase （LCHAD）is a disease whose incidence is approximately 3 cases/100 000 births, with autosomal recessive inheritance.

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from saline strain Idiomarina loihiensis

Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.

Separation and Purification of GST-glycerol-3-phosphate Dehydrogenase

In order to investigate the expression of glycerol-3 -phosphate dehydrogenase by GCY1 gene in recombinant Saccharomyces cerevisiae, induction culture of the S. cerevisiaestrain was performed with SD-URA 2% galactose, 3 × YP ＋ 6% glucose, SC-URA 2% galactose, and SC-URA 2% galactose ＋ 5% NaCI glyeerol-3-phosphate dehydregenase, the cultured S. cerevisiaewas comminuted followed by full-automatic high-speed purification, and SDS-PAGE gel electrophoresis was performed for molecular weight of the GST fusion protein. The results showed that after shaking culture of the S. cerevisiae containing GCY1 at 25 ℃, the OD values of its 3 × YP ＋ 6% glucose culture and SC-URA 2% galaetose ＋ 5% NaC1 culture were 8.75 and 7.35, respectively. It was shown by purification with a Profinia low-pressure liquid chromatograph that only the S. cerevisiae cultured in SC-URA 2% galactose ＋ 5% NaC1 medium expressed glycerel-3-phosphate de- hydrogenase, the molecular weight of which was detected as 65 ku by SDS-PAGE gel electrophoresis.

Role of Cathepsin G in the Degradation of Glyceraldehyde-3-Phosphate Dehydrogenase Triggered by 4-Hydroxy-2-Nonenal in U937 Cells

Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.

Increased Expression of 11<i>β</i>-Hydroxysteroid Dehydrogenase Type 1 in Experimental Periodontitis Induced by Lipopolysaccharide from <i>Porphyromonas gingivalis</i>

It has been proposed that 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which activates glucocorticoids, plays a role in chronic inflammatory diseases including metabolic diseases, rheumatoid arthritis, and ulcerative colitis. We have recently reported that the expression of 11β-HSD1 is increased in the gingiva of patients with chronic periodontitis and in that of rats with ligature-induced periodontitis. In this study, to further demonstrate the involvement of 11β-HSD1 in chronic periodontitis, the expression of 11β-HSD1 was investigated in another rat model of experimental periodontitis induced by intragingival injection of lipopolysaccharide from Porphyromonas gingivalis (LPS-PG). Alveolar bone loss was observed two weeks after intragingival injection of LPS-PG. The level of 11β-HSD1 mRNA assessed by real-time reverse transcriptase-polymerase chain reaction was significantly elevated in LPS-PG-induced periodontitis compared with controls. The expression of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which inactivates glucocorticoids, was not significantly different between control and LPS-PG-induced periodontitis. The expression of 11β-HSD1 was significantly correlated with that of TNF in LPS-PG-induced periodontitis. The increased expression of 11β-HSD1 protein in LPS-PG-induced periodontitis was confirmed by immunohistochemistry using anti-11β-HSD1 antibody. These results further suggest a role for 11β-HSD1 in the pathogenesis of chronic periodontitis.

Interceptive Activities of Some New 3β─Hydroxysteroid Dehydrogenase Inhibitors

Fourteen compounds of azastene and epostane derivatives (from YG101 to YG114) have been studied. Results showed that only YG102 and YG103 wore found to be positive in interceptivo activities, although they were less potent than their parent compound──azastene. Levels ofprogesterone in plasma were decreased significantly after administrstion or YG102, 103 and 106. Only YG107 possessed an interceptive activity approximately as potent as that of its paront compound──epostane. Epostane is a mixture of its enol and keto forms and the percentage of both forms defends on various condions. Since YG107 exists only in one form, we believe this derivative of epostane might be useful in the future work.

Use of a Simple Fabrication Process to Produce a Biosensor： The 3-Hydroxybutyrate Dehydrogenase Case

This study was aimed to construct a biodegradable but reliable 3-β-hydroxybutymte biosensor. In this context a versatile paper based biosensor, quickly, easily and cheaply fabricated is reported. The procedure of fabrication is based on the assumption that the introduction of the enzyme in the carbon ink will allow enzyme stabilization and facilitate the study of the catalysis of enzymes and the detection of substrates. To prove this concept we use the enzyme 3-hydroxybutyrate dehydrogenase, in aqueous solution. This enzyme was chosen because it catalyzes the 3-β-hydroxybutyrate, which results from ketoacidosis. The quantification this substance in the diabetics＇ blood is very important as it can increase the reliability of the diagnosis of glycaemia. To prove the multi-use of this biosensor we not only study the redox process in steady state and during the catalytic process, but also detected and quantify the 3-β-hydroxybutyrate. Our results showed that it was possible to study the redox process that occurred during the catalysis and to confirm the amino acid residues that participate in it. It was also observed that glucose and ascorbic acid can interfere in the detection and quantification of the 3-β-hydroxybutyrate, what should be in mind when the quantification of the 3-β-hydroxybutyrate is made in blood samples.

Choline dehydrogenase interacts with SQSTM1 to activate mitophagy and promote coelomocyte survival in Apostichopus japonicus following Vibrio splendidus infection

Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes,leading to the production of excessive reactive oxygen species(ROS)and irreversible apoptotic cell death.Emerging evidence suggests that mitochondrial autophagy(mitophagy)is the most effective method for eliminating damaged mitochondria and ROS,with choline dehydrogenase(CHDH)identified as a novel mitophagy receptor that can recognize non-ubiquitin damage signals and microtubule-associated protein 1 light chain 3(LC3)in vertebrates.However,the functional role of CHDH in invertebrates is largely unknown.In this study,we observed a significant increase in the mRNA and protein expression levels of A.japonicus CHDH(AjCHDH)in response to V.splendidus infection and lipopolysaccharide(LPS)challenge,consistent with changes in mitophagy under the same conditions.Notably,AjCHDH was localized to the mitochondria rather than the cytosol following V.splendidus infection.Moreover,AjCHDH knockdown using si RNA transfection significantly reduced mitophagy levels,as observed through transmission electron microscopy and confocal microscopy.Further investigation into the molecular mechanisms underlying CHDH-regulated mitophagy showed that AjCHDH lacked an LC3-interacting region(LIR)for direct binding to LC3 but possessed a FB1 structural domain that binds to SQSTM1.The interaction between AjCHDH and SQSTM1 was further confirmed by immunoprecipitation analysis.Furthermore,laser confocal microscopy indicated that SQSTM1 and LC3 were recruited by AjCHDH in coelomocytes and HEK293T cells.In contrast,AjCHDH interference hindered SQSTM1 and LC3 recruitment to the mitochondria,a critical step in damaged mitochondrial degradation.Thus,AjCHDH interference led to a significant increase in both mitochondrial and intracellular ROS,followed by increased apoptosis and decreased coelomocyte survival.Collectively,these findings indicate that AjCHDH-mediated mitophagy plays a crucial role in coelomocyte survival in A.japonicus following V.splendidus infection.

puuC基因过表达对肺炎克雷伯氏菌发酵甘油生产3-HP的影响

为获得高产3-羟基丙烯酸(3-hydroxypropionic acid,3-HP)的基因重组菌,从肺炎克雷伯氏菌(Klebsiella pneumoniae,K.pneumoniae)HD79中克隆乙醛脱氢酶基因puuC,进行同源过表达,探究其对K.pneumoniae HD79甘油还原途径中乙醛脱氢酶(Aldehyde dehydrogenase,ALDH)活性与3-HP产量的影响。结果表明,成功构建了同源过表达puuC的重组菌株K.pneumoniae HD79-T,过表达puuC基因后提高了K.pneumoniae HD79的3-HP生产能力。K.pneumoniae HD79-T的3-HP最高产量为(2.05±0.03)g·L^(-1),比K.pneumoniae HD79((1.47±0.02)g·L^(-1))提高了39.46%。K.pneumoniae HD79-T中puuC基因、甘油脱水酶基因(dhaB)和1,3-丙二醇氧化还原酶基因(dhaT)的相对表达量较K.pneumoniae HD79分别提高94.35、1.72和1.35倍。K.pneumoniae HD79-T中ALDH、甘油脱水酶(Glycerol dehydratase,GDHt)和1,3-丙二醇氧化还原酶(1,3-propanediol oxidoreductase,1,3-PDOR)的酶活性相比,K.pneumoniae HD79分别提高了27.40%、18.85%和5.10%。本研究构建的K.pneumoniae HD79-T提高了甘油还原途径关键酶ALDH的酶活性,是高效利用甘油发酵生产3-HP的前景菌株。

外源添加VB_(12)对肺炎克雷伯氏菌代谢3-羟基丙酸的影响

为了探究外源添加维生素B12(Vitamin B12,VB_(12))对Klebsiella pneumoniae HD79和K.pneumoniae HD79-T利用甘油还原途径生产3-羟基丙酸(3-hydroxypropionic acid,3-HP)的影响以及摸索VB_(12)对K.pneumoniae HD79和K.pneumoniae HD79-T的阈值上限,将不同浓度(0.01、0.02、0.03、0.04、0.05 g/L)的VB_(12)添加到K.pneumoniae HD79及K.pneumoniae HD79-T的发酵培养基中,利用HPLC检测其底物消耗及产物产生情况、qRT-PCR检测还原途径相关基因的mRNA表达情况以及酶联免疫试剂盒检测代谢相关酶活性。结果表明,VB_(12)对K.pneumoniae HD79和K.pneumoniae HD79-T的阈值为0.01 g/L和0.03 g/L。与未添加VB_(12)相比,菌株K.pneumoniae HD79和K.pneumoniae HD79-T的3-HP产量分别提高了24.39%,8.86%;醛脱氢酶基因puuC表达量分别提高了2.49倍和1.68倍;ALDH、GDHt和PDOR的酶活力分别提高了50.24%、40.36%和18.29%,及30.49%、37.84%和13.56%。说明通过外源添加辅酶因子VB_(12)对肺炎克雷伯氏菌高产3-HP是可行策略。

Limonin inhibits the stemness of cancer stem-like cells derived from colorectal carcinoma cells potentially via blocking STAT3 signaling

BACKGROUND Limonin is one of the most abundant active ingredients of Tetradium ruticarpum.It exerts antitumor effects on several kinds of cancer cells.However,whether limonin exerts antitumor effects on colorectal cancer(CRC)cells and cancer stem-like cells(CSCs),a subpopulation responsible for a poor prognosis,is unclear.AIM To evaluate the effects of limonin on CSCs derived from CRC cells.METHODS CSCs were collected by culturing CRC cells in serum-free medium.The cytotoxicity of limonin against CSCs and parental cells(PCs)was determined by cholecystokinin octapeptide-8 assay.The effects of limonin on stemness were detected by measuring stemness hallmarks and sphere formation ability.RESULTS As expected,limonin exerted inhibitory effects on CRC cell behaviors,including cell proliferation,migration,invasion,colony formation and tumor formation in soft agar.A relatively low concentration of limonin decreased the expression stemness hallmarks,including Nanog andβ-catenin,the proportion of aldehyde dehydrogenase 1-positive CSCs,and the sphere formation rate,indicating that limonin inhibits stemness without presenting cytotoxicity.Additionally,limonin treatment inhibited invasion and tumor formation in soft agar and in nude mice.Moreover,limonin treatment significantly inhibited the phosphorylation of STAT3 at Y705 but not S727 and did not affect total STAT3 expression.Inhibition of Nanog andβ-catenin expression and sphere formation by limonin was obviously reversed by pretreatment with 2μmol/L colievlin.CONCLUSION Taken together,these results indicate that limonin is a promising compound that targets CSCs and could be used to combat CRC recurrence and metastasis.

结直肠癌组织PHGDH、APOL3表达及临床预后价值

目的研究结直肠癌(CRC)中3-磷酸甘油酸脱氢酶(PHGDH)、载脂蛋白L3(APOL3)表达,分析两者对CRC患者的预后评估价值。方法回顾性收集2018年3月—2021年2月首都医科大学大兴教学医院消化内科收治的CRC患者112例临床资料。采用实时荧光定量PCR(qPCR)和免疫组织化学法检测癌组织及癌旁组织中PHGDH、APOL3 mRNA和蛋白表达;Kaplan-Meier法分析PHGDH、APOL3 mRNA表达对CRC患者生存预后的影响;Cox回归分析CRC预后的影响因素;受试者工作特征(ROC)曲线评价PHGDH、APOL3 mRNA表达水平对CRC患者预后的评估价值。结果CRC患者癌组织中PHGDH mRNA相对表达量高于癌旁组织,APOL3 mRNA相对表达量低于癌旁组织(t/P=52.982/<0.001,35.679/<0.001);CRC患者癌组织中PHGDH、APOL3蛋白阳性率分别为78.10%(90/112)、8.57%(10/112),与癌旁组织的7.62%(8/112)、76.19%(88/112)比较,差异有统计学意义(χ^(2)/P=123.722/<0.001,110.367/<0.001);TNM分期Ⅲ期、有淋巴结转移的CRC癌组织中PHGDH mRNA表达水平高于TNM分期Ⅰ~Ⅱ期、无淋巴结转移,而APOL3 mRNA表达水平低于TNM分期Ⅰ~Ⅱ期、无淋巴结转移(PHGDH:t/P=52.268/<0.001,51.315/<0.001;APOL3:t/P=15.873/<0.001,14.769/<0.001)。PHGDH mRNA高表达组3年总生存率为50.00%(26/52),低于低表达组的73.33%(44/60)(Log rankχ^(2)=4.169,P=0.041);APOL3 mRNA低表达组3年总生存率为46.30%(25/54),低于高表达组的77.59%(45/58)(Log rankχ^(2)=10.650,P=0.001)。TNM分期Ⅲ期、淋巴结转移、PHGDH mRNA高为影响CRC患者预后的独立危险因素[OR(95%CI)=1.465(1.172~1.832),1.501(1.159~1.944),1.384(1.088~1.761)],APOL3 mRNA高为独立保护因素[OR(95%CI)=0.775(0.617~0.924)];PHGDH、APOL3 mRNA表达水平及二者联合评估CRC患者预后的曲线下面积(AUC)分别为0.830、0.816、0.922,二者联合的AUC大于PHGDH、APOL3 mRNA表达水平单独评估(Z=4.482、4.130,P均<0.001)。结论CRC中PHGDH表达上调,APOL3表达下调,在CRC中均发挥重要的促癌作用,联合检测PHGDH、APOL3的表达有助于评估CRC的预后。

Fe^(3+)对活性污泥系统的影响

通过小试研究了微量Fe3+对活性污泥系统的影响。将浓度为3 mg／L,5 mg／L,10 mg／L,20 mg／L,30 mg／L,50 mg／L和80mg／L的Fe3+分别投加到活性污泥系统中,反应4 h后测定系统出水COD、活性污泥的SVI、脱氢酶活性及其EPS组分。结果表明,Fe3+浓度小于50 mg／L时对活性污泥的脱氢酶活性具有促进作用,浓度为10 mg／L时促进作用最强;Fe3+浓度在80 mg／L以下均具有良好的絮凝作用,浓度在30 mg／L以下时絮凝作用最强。两种作用的共同结果影响系统对COD的去除效果。对活性污泥EPS组分的测定表明,Fe3+的絮凝作用对SVI的影响是主要的。

qa-3稀有密码子和mRNA结构改造及其在大肠杆菌中的高效表达

奎尼酸脱氢酶的高表达是奎尼酸生物合成的代谢工程研究的关键和基础。粗糙脉孢菌基因组中编码奎尼酸脱氢酶的基因qa-3在大肠杆菌中不表达,根据大肠杆菌密码子使用频率分析qa-3基因,发现有27个稀有密码子,其中编码Arg(R)的有8个,编码Gly(G)的有9个。编码精氨酸的AGG(AGA)两个稀有密码子紧密相连(R区),另外还有个相对比较集中的GGG密集区G区。进一步通过计算机分析其qa-3基因mRNA二级结构,发现改变5′和3′末端个别碱基对其二级结构的影响很大,可以使mRNA的自由能由野生型的-374.3kJ/mol降低到最小-80.5kJ/mol,从而大大减少mRNA两端二级结构的产生,而仅仅改变R区和G区的稀有密码子自由能变化很小。通过对该基因密码子改造和优化5′和3′末端对其mRNA二级结构的影响,在大肠杆菌中表达真菌的基因qa-3,并测到了奎尼酸脱氢酶活性,为构建产奎尼酸工程菌株奠定基础。